Current data suggest that HIV-1 vaccine candidates that induce both a strong and balanced Thl/Th2 immune response will facilitate the generation of both cytotoxic T-cell responses as well as neutralizing antibodies. Preclinical studies have shown that either of these effector mechanisms alone can protect macaques, suggesting that both together will result in more effective immunity to HIV-I. However, efforts to generate broad neutralizing antibodies using envelope immunogens and conventional protocols have to date been unsuccessful. Using immune stimulating complexes (ISCOMs) we have previously demonstrated that both potent cellular as well as humoral immune responses to HIV-1 antigens could be generated, and were effective in protecting rhesus monkeys from SHIV infection. However, the use of monovalent gp120 to prime responses resulted in protection which was limited in breadth. This project is specifically focused to investigate the optimal prime-boost strategy capable of generating the broadest possible neutralizing antibody responses. In close collaboration with project 1, we will evaluate the best Env antigenic structure(s) to most effectively prime the humoral response. In the first year, several different primary isolate clade B sf162 Env Ags (gp140s with and without V-deletes or +/- receptor complexes) will be compared for the best ability to prime the boost with a pool of well characterized peptide/mimotopes (formulated in ISCOMS). Once the optimal clade B Ag structure which gives optimal priming is identified, then the potential benefit of multivalent Env ISCOM immunization using envelope antigens from different clades (C and E) will be examined. Concurrently efforts to improve the "boost" will be undertaken by further characterizing and adding phages expressing mimotopes, some of which are specific for the broad cross-neutralizing monoclonal antibodies such as 2G12, 2F5, and B12. The potent T-helper responses elicited by ISCOMs will be used to elicit high titer antibodies in rodents and primates, and to evaluate their ability to neutralize a panel of primary isolates. Once the lead prime-boost strategy has been identified based on broad neutralizing activity of primary isolates, then Gag and Pol will be added to the vaccine regimen to recruit helper and CTL responses to multiple targets. The efficacy of these vaccine candidates will be evaluated by then challenging these macaques with heterologous pathogenic SHIV variants by systemic and mucosal routes. All immunogens will be formulated in ISCOMs based on refined components approved for human clinical trials.